Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification.
The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)
Suzuki
H
H
Department of Biophysical Chemistry, Kitasato University School of Medicine.
Mukouyama
E B
EB
Kamei
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology*; D006868:Hydrolysis; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000494:pharmacology
D002250:Carbon Radioisotopes; D015852:Eukaryotic Initiation Factor-2; D013438:Sulfhydryl Compounds; D006153:Guanosine Diphosphate; D014357:Trypsin; D005033:Ethylmaleimide